Disassembly of the Mu Transposase Tetramer by the Clpx Chaperone

Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Disassembly of the Mu transposase tetramer by the ClpX chaperone Igor Levchenko, Li Luo, and Tania A. Baker Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, 68-523, Cambridge, Massachusetts 02139 USA Mu transposition is promoted by an extremely stable complex containing a tetramer of the transposase (MuA) bound to the recombining DNA. Here we purify the Escherichia coli ClpX protein, a member of a family of multimeric ATPases present in prokaryotes and eukaryotes (the Clp family), on the basis of its ability to remove the transposase from the DNA after recombination. Previously, ClpX has been shown to function with the ClpP peptidase in protein turnover. However, neither ClpP nor any other protease is required for disassembly of the transposase. The released MuA is not modified extensively, degraded, or irreversibly denatured, and is able to perform another round of recombination in vitro. We conclude that ClpX catalyzes the ATP-dependent release of MuA by promoting a transient conformational change in the protein and, therefore, can be considered a molecular chaperone. ClpX is important at the transition between the recombination and DNA replication steps of transposition in vitro; this function probably corresponds to the essential contribution of ClpX for Mu growth. Deletion analysis reveals that the sequence at the carboxyl terminus of MuA is important for disassembly by ClpX and can target MuA for degradation by ClpXP in vitro. These data contribute to the emerging picture that members of the Clp family are chaperones specifically suited for disaggregating proteins and are able to function with or without a collaborating protease. [Key Words: Clp; Hspl04; transposition; phage Mu; replication] Received }uly 17, 1995; revised version accepted August 22, 1995. Higher order protein-DNA complexes are often critical genome. This tetramer pairs the two ends of the Mu intermediates in initiation of transcription, recombina DNA, cleaves these ends, and joins the cleaved ends to a tion, and replication. Timely assembly and disassembly new DNA site in a reaction called strand transfer of these complexes is likely to be essential for the proper (Craigie and Mizuuchi 1987; Surette et al. 1987, 1991; function and regulation of these processes. The protein- Lavoie et al. 1991; Mizuuchi et al. 1992). A second Mu- DNA complexes involved in site-specific recombination encoded transposition protein, MuB, participates in and transposition are among the best understood and, strand transfer by activating MuA and delivering an in- therefore, useful for dissecting the general principles termolecular target site to the transposase complex (Ad- governing the assembly, organization, and disassembly zuma and Mizuuchi 1988; Baker et al. 1991; Surette and of such complexes. Chaconas 1991). Biochemical analysis of transposition by elements as Transposition is used for two steps in the phage Mu diverse as phage Mu, TnlO, Tn7, and human immuno life cycle: (1) integration of the Mu genome into that of deficiency virus (HIV) indicate that the transposase and the host cell during infection and (2) replicative ampli integrase proteins act by a similar mechanism (for re fication of the DNA during lytic growth. Although it is view, see Mizuuchi 1992) and function in stable multi well established that phage Mu uses transposition to rep meric complexes (Surette et al. 1987; Haniford et al. licate its genome, how replication is initiated after the 1991; Bainton et al. 1993; Ellison and Brown 1994). Al strand transfer reaction is not well understood. Mu rep though Mu transposase and HIV integrase have only a lication in vivo requires several essential Escherichia modest degree of amino acid sequence similarity (Baker coh genes that encode components of the host replica and Luo 1994), recent determination of the structures of tion machinery, indicating that Mu replication forks are the core domains of these proteins reveal remarkable similar to those used for chromosomal replication (Tous- similarities surrounding the active sites (Dyda et al. saint and Faelen 1974; Toussaint and Resibois 1983; 1994; Rice and Mizuuchi 1995). Resibois et al. 1984; Ross et al. 1986). Strand transfer Mu transposase (MuA) catalyzes the DNA cleavage complexes (STC) can be replicated efficiently in E. coli and joining reactions central to recombination. MuA is extracts made from uninfected cells indicating that no monomeric in solution but forms a stable tetramer upon Mu proteins other than MuA and MuB are required (Mi binding to specific sequences at each end of the phage zuuchi 1983), although the Mu arm functions stimulate GENES & DEVELOPMENT 9:2399-2408 © 1995 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/95 $5.00 2399 Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Levchenko et al. replication in vivo (Waggoner et al. 1981). Recently^ in joined by strand transfer. The releasing activity in the vitro replication with a more purified system confirmed extract was fractionated by chromatography; one activ roles for eight of the E. coli replication proteins and in ity behaved as a single component and was purified ex dicated that additional host factors are required (Krukli- tensively (see Materials and methods). The peak of re tis and Nakai 1994). leasing activity correlated with a 46-kD protein during In addition to the replication enzymes, the E. coli clpX several chromatography steps; the activity of fractions gene product is required for lytic growth of phage Mu from the penultimate column (Mono Q) is shown (Fig. (Mhammedi-Alaoui et al. 1994). The kinetics of the la,b). After Superose 6 chromatography, this 46-kD pro block in phage growth after induction of a lysogen indi tein was >90% pure (Fig. la). cate that ClpX functions after the first strand transfer Several lines of evidence established that the 46-kD reaction but before onset of extensive replication protein was the ClpX protein. The protein (1) reacted (Mhammedi-Alaoui et al. 1994). ClpX is a member of a efficiently with anti-ClpX antibody on a Western blot conserved family of ATPases (the Clp family) present in (data not shown); (2) supported degradation of a known prokaryotes and eukaryotes (Gottesman et al. 1990, substrate of the ClpXP protease in the presence of ClpP 1993). Many organisms, including E. coli have multiple and ATP (see below); and (3) was more abundant in ex family members, many of which are heat shock proteins. tracts made from cells overproducing ClpX than from The best studied Clp protein, E. coli ClpA, forms a com nonoverproducing cells. Active fractions of ClpX puri plex with the ClpP protein, a small serine protease (un fied from overproducing cells were >95% pure as judged related in sequence to the other Clp proteins) to promote by densitometery of a Coomassie Blue-stained SDS gel ATP-dependent degradation of specific proteins (Hwang (Fig. Ic). Release of MuA from the STC by purified ClpX et al. 1988; Katayama et al. 1988). ClpX was first purified was relatively efficient; in a reaction containing 1.3 based on its ability to degrade, with the ClpP protease, pmoles of MuA, 1.7 to 3.3 pmoles of ClpX gave rise to the replication initiator protein of phage k, XO protein protein-free product DNA within 5 min (data not (Wojtkowiak et al. 1993). However, although Mu growth shown). requires ClpX and some forms of the Mu repressor ap The ATP requirement for release of MuA from the pear to be degraded by ClpXP (Geuskens et al. 1992), Mu STC by ClpX was investigated by modifying the reaction propagates relatively normally in cipP-deficient cells (Mhammedi-Alaoui et al. 1994), indicating that the es sential role of ClpX in Mu growth does not involve pro tein degradation by ClpXP. In this study, we purify a factor from E. coli cell ex MonoQ. fractions Superose 6 overproduced ClpX tracts on the basis of its ability to displace the MuA tetramer from the DNA after strand transfer. This factor is the ClpX protein. ClpX catalyzes the ATP-dependent disassembly of the MuA tetramer into monomers with out detectable degradation and the released protein is active in another round of recombination. The impor tance of ClpX for Mu replication in vitro is also demon strated. Implications of these data for the pathway of Mu replication and the mechanism of action of the Clp pro tein family are discussed. Results Purification of ClpX protein as a factor able to release MuA from the STC Upon completion of the cleavage and strand transfer re actions in vitro, MuA remains bound to the recombined Figure 1. ClpX removes MuA from strand transfer complexes. DNA in a protein-DNA complex called the STC or type [a] SDS-PAGE of ClpX fractions after Mono Q [left] and Super II transposome. Although noncovalent, this complex is ose 6 {right) chromatography. Arrow indicates ClpX protein. stable for days and resists treatment with 6 M urea and Prestained protein markers are myosin (h-chain) 214 kD, phos- heating to 65°C (Surette et al. 1987). Factors able to re phorylase Bill kD, BSA 74 kD, ovalbumin 45 kD, and carbonic move MuA from the STC were detected in crude E. coli anhydrase 29 kD. [b] MuA releasing activity of Mono Q frac tions of ClpX (5 |xl of each fraction [0.2-1.0 (x,g of total protein] extracts active in Mu DNA replication (data not shown). was added) assayed by agarose gel electrophoresis; arrow indi One assay used to detect removal of MuA from the DNA cates STC, bracket indicates topoisomers of free strand transfer depended on the difference in mobility between the STC DNA. The protein-DNA complexes and free DNA products and the strand transfer products on a native agarose gel.

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